WO2002052029A1

WO2002052029A1 - Process for producing recombinant protein and fused protein

Info

Publication number: WO2002052029A1
Application number: PCT/JP2001/011438
Authority: WO
Inventors: Masahiro Furutani; Junichi Hata; Akiko Togi
Original assignee: Sekisui Chemical Co., Ltd.; Marine Biotechnology Institute Co., Ltd.
Priority date: 2000-12-26
Filing date: 2001-12-26
Publication date: 2002-07-04
Also published as: CA2432716A1; US7276355B2; KR20030065574A; US20080081361A1; AU2002217505B2; EP1354959A1; JPWO2002052029A1; EP1354959B1; US7608424B2; JP4038555B2; US20040146969A1; WO2002052029A9; ATE463580T1; EP1354959A4; KR100892889B1; DE60141769D1

Abstract

It is intended to provide an expression system of a recombinant protein and a cell-free translation system with the use of a host wherein a target protein is surely incorporated into the stereo structure of chaperonin as a fused protein with a chaperonin subunit (namely, a molecular chaperonin of about 60 kDa, heat shock protein of 60 kDa or thermosome) to thereby inhibit the expression of toxicity of the target protein in the host, the formation of an enclosed matter, and digestion with a protease, thereby universally expressing any protein as a soluble protein. Namely, a process for producing a protein characterized by transcribing and translating a gene containing a gene encoding a chaperonin subunit and a gene encoding a target protein to thereby synthesize a fused protein in which the target protein is linked to the chaperonin subunit via a peptide bond.

Description

Specification

TECHNICAL FIELD The present invention relates to a method for producing a recombinant protein and a fusion protein.

The present invention enables the production of a protein that has been difficult to synthesize as an active protein in the expression of a recombinant protein in a host system or in the expression of a protein in a cell-free translation system. The present invention relates to a novel protein production method and a fusion protein for realizing protein synthesis and purification. Background art

To date, recombinant protein expression systems and cell-free translation systems have been established in many hosts such as bacteria, yeast, insects, animal and plant cells, and transgenic animals and plants. In particular, recombinant protein production by mammalian cultured cells is becoming a standard system for the production of therapeutic drugs because of appropriate post-translational modifications. However, the biotechnology industry, which has a lower level of protein synthesis and requires larger culture tanks than microbial host systems, and handles new drugs, is expected to lack production facilities in the future (Garber, K. , 2001, N at. Biotec h. 19,

1 8 4—1 8 5). In recent years, protein production technology using transgenic animals and plants, whose production efficiency has been improved, has not yet gained the full range of reliability (Garber, K., 201, N at. Biotec. 1 9, 1 84—1 8 5). On the other hand, it is often difficult to obtain a large amount of active protein in the above-mentioned recombinant protein expression system developed so far. If the target protein has any toxicity to the host, the synthesis of the protein is suppressed and the expression level is reduced. In addition, even when the target protein is expressed as a soluble protein, it may be degraded by the host protease, resulting in extremely low production. Furthermore, even when the target protein is expressed, folding may not be successful, and an inclusion body may be formed. In this case, even if the protein is solubilized and refolded, the amount of the active protein finally obtained is extremely small. In particular, when a cell-free translation system is used, inclusion bodies are easily formed. If inclusion bodies are formed, the solution can be, for example, daltathione-S-transferase (GST) (Smith, DB, eta 1.., 1988, Gene 6.7, 31.40) , Thioredoxin (LaVallie, ER etal., 1993, Bio / echechnology 11, 187-18), maltose-binding protein (Guan, C., eta1., Gene 67, 21-1-30), etc., but a method of expressing it as a fusion protein has been used, but the formation of inclusion bodies was rarely eliminated with high efficiency. In addition, a method of co-expressing a target protein with a molecular chaperone, which is a group of proteins that support the protein folding reaction, to increase the expression level of the target protein in a soluble fraction (Nishiharaeta 1.1998, Apply) Environ. Microbiol., 6 4, 16 9 4-16 9 9), but the current situation is that the amount of active protein has not been increased dramatically.

As a solution to the degradation of the target protein by the host protease, for example, in E. coli, a host (Phillipsetal. A method using B acteriol. 159, 283-287) has been devised, but it is rarely possible to avoid the effects of protease-induced degradation. The problem could not be solved fundamentally. As described above, conventional protein expression techniques have major problems such as toxicity to the host, degradation by host protease, and inclusion body formation.The amount of expression differs significantly depending on the type of protein to be expressed. Consequently, it was necessary to examine the expression conditions for each protein by trial and error. For this reason, there has been a demand for the development of technology that fundamentally solves the above problems. Summary of the Invention

In view of the above, the present invention provides a method for reliably transforming a target protein into a fusion protein with a shanin nin subunit, that is, about 60 kDa molecular chaperone, heat shock protein 60 kDa, or a thermosome. By putting the protein into the three-dimensional structure of the mouth nin, the expression of the target protein's toxicity to the host, the formation of inclusion bodies, and the protease It is an object of the present invention to provide a recombinant protein expression system and a cell-free translation system using a host capable of suppressing degradation and capable of universally expressing any protein as a soluble protein in large amounts.

The present invention transcribes and translates a gene containing a gene encoding a spermulin ninsubunit and a gene encoding a target protein, and links the target protein to the sharonin subunit via a peptide bond. A method for producing a protein, comprising synthesizing a fusion protein.

The fusion protein is composed of 1 to 20 linked mouth ninsubunits linked to each other, the N-terminal of the linked mouth ninth subunit, the C-terminus of the linked mouth ninsubut, or It preferably comprises a target protein linked via a peptide bond to the junction between the oral nin subunits.

In the present invention, the gene containing the gene encoding the ninja ninsubunit and the gene containing the gene encoding the target protein are used in two different plasmids capable of coexisting and replicating in the same host. May be introduced and co-expressed in the same host, or may encode only the gene encoding the Shanin subunit and the gene containing the gene encoding the target protein, and encode only the Shanin mouth The gene may be introduced into two different plasmids capable of coexisting and replicating in the same host, and co-expressed in the same host.

The above-mentioned fusion protein is preferably one in which the target protein is stored inside the Shako Nin ring in a state of being linked to the Shako Nin subunit via a peptide bond.

The chaperonin ring may form a two-layer structure non-covalently associated via a ring surface, or a non-covalently linked fibrous structure via a ring surface or a side surface thereof. May be formed.

In the present invention, there is provided a step of providing a cleavage sequence for a limited-degradable protease at the junction between the ninth subunit and the target protein, and cleaving the target protein from the fusion protein using the limited-degradable protease. It may be. At this time, it is preferable to provide a cleavage sequence for the limited-degradation type protease also at the junction between the shark mouth nin subunits. In the present invention, a method may be provided in which a methionine residue is provided at the junction between the ninnin subunit and the target protein, and the target protein is cleaved from the fusion protein by CNBr.

In the present invention, bacteria, archaea, eukaryotes, and the like can be mentioned as organisms from which Shanin ninth is derived.

In the present invention, the fusion protein may be synthesized in any host of bacteria, yeast, animal cells, plant cells, insect cells, animal individuals, plant individuals, or insect individuals, or a cell-free translation system. May be used to synthesize the fusion protein.

In the present invention, the gene encoding the target protein is preferably a partial gene encoding an amino acid sequence of 6 or more residues of mammalian-derived cDNA or mammalian-derived cDNA.

In the present invention, the target protein may be a heavy chain of a mammal-derived antibody, a light chain of a mammal-derived antibody, or a full-length FV region single-chain antibody of a mammal-derived antibody, or six or more residues thereof. Partial proteins: viral antigens, seven-transmembrane receptor proteins, and site proteins.

According to the present invention, there is provided a fusion protein consisting of a spore opening ninsubunit and a target protein, wherein the target protein is linked to the sway opening subunit via a peptide bond, and a Thus, a fusion protein stored inside is obtained. The obtained fusion protein is also one of the present invention.

The shaved mouth ring may form a two-layer structure non-covalently associated via a ring surface, or a fibrous non-covalently connected ring surface or a side surface thereof. A structure may be formed. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a diagram schematically showing the three-dimensional structure of Escherichia coli Shaguchinin (GroEL).

Fig. 2 is a diagram showing an example of the design of a fusion protein consisting of archaeonin derived from archaea having 8 subunits and a target protein.

Figure 3 shows the restriction map of the expression vector pETD (TCPi3) n (n = 1 to 4). FIG.

FIG. 4 is a diagram showing the results of SDS-PAGE performed on (TCP jS) n (n = l to 4) and a fusion protein of a TCP tetramer and a target protein.

FIG. 5 is a diagram showing the results of observation of a TCP 3 linked product by a transmission electron microscope. FIG. 6 is a diagram showing the results of Western blotting in Example 5 _c FIG. 7 is a diagram showing the results of Western plotting in Example 7 _c FIG. 8 is a diagram showing the expression vector p T r ( It is a figure which shows the restriction map of GroE) n (n = l-7).

FIG. 9 is a diagram showing the results of SDS-PAGE performed on the soluble fraction of Escherichia coli expressing the Escherichia coli Shaguchinin GroEL conjugate in Example 8. Detailed Disclosure of the Invention

Hereinafter, the present invention will be described in detail.

In the present invention, the target protein and the gene encoding the target protein are used by using a gene containing the gene encoding the target protein and the gene containing the gene encoding the target protein (hereinafter, also referred to as a gene encoding the fusion protein). Produces a fusion protein consisting of units.

When the cell is subjected to stress such as heat shock, it exerts support on protein folding in the presence or absence of ATP, an energy substance, and contributes to structural stability. In general, the expression of a group of proteins called molecular chaperones is induced. Among these molecular chaperones, those whose subunits have a molecular weight of about 6 kDa are bacteria, archaea, and eukaryotes. It is present in all living organisms and has functions of supporting protein folding and preventing degeneration.

Shanin is composed of 14 to 18 subunits and has a three-dimensional structure consisting of two layers of rings (hereinafter, the ring is referred to as the Shanin ring). It has a cavity (cavity) with a height of 5 nm and a height of 14.5 nm (see Fig. 1). The cavities of the further Ninring have enough space for one 60 kDa spherical protein. This protein has the function of temporarily storing folding intermediates and denatured proteins of various proteins in this cavity. When a quality fold is formed, it releases the stored protein from the cavity, coupled with ATP degradation. Bacterial and archaeal shadinin can be easily mass-produced in the cytoplasmic soluble fraction of E. coli while maintaining the ring structure. This suggests that various Shanins of different origins also self-assemble in Escherichia coli and take a two-layered ring structure of 14 to 18 mer.

According to the three-dimensional structure of Shahkonin, which was revealed by X-ray crystal structure analysis, the N-terminal and C-terminal of Shahkonin's subunit are both located on the cavity side, resulting in a highly flexible structure. . In particular, at least the 20-amino acid at the C-terminus shows a highly flexible structure (George et al., 2000, Cell, 100, p. 561-573).

The chaperone used in the present invention is not particularly limited, and any of bacteria, archaebacteria, and eukaryotes can be used. In addition, in the present invention, not only wild-type but also amino acid mutants can be used as long as the ability of self-assembly of Shahkonin to the ring structure is maintained. For example, when a mutant having a reduced association power of each subunit of Shaguchinin is used, it is easier to recover the stored target protein.

The protein of interest in the present invention is not particularly limited, and all disease-related gene products derived from higher animals such as humans and mice and a group of enzymes effective for chemical processes can be the protein of interest. For example, hepatitis B virus Proteins (viral antigens), such as coat proteins, core proteins, proteases, reverse transcriptase, integrase, etc., encoded by pathogenic virus genomes such as hepatitis C virus, HIV, and influenza; Chain, light chain of mammal-derived antibody, full length of FV region single-chain antibody (sc FV) of mammal-derived antibody, or partial protein of 6 or more residues thereof, Fab, (Fab) 2, and Therapeutic antibody that is a complete antibody type; diagnostic antibody; seven-transmembrane receptor (G protein-coupled receptor); platelet growth factor, blood stem cell growth factor, hepatocyte growth factor, transformin Growth factors, nerve growth. Nutrition factors, fibroblast growth factors, growth factors such as insulin-like growth factor; tumor death factors, interferon, interleukin, erythropoietin, granulocyte colony stimulating factor, macrophage ■ colony stimulating factor, Site power-ins such as albumin and human growth hormone I can do it.

The structure of Shachinin differs depending on the organism and tissue. In the case of bacteria, mitochondria and chloroplasts, the number of subunits constituting the spheronin ring is 7, whereas in the case of eukaryotic cytoplasm and archaea, the number of subunits is large. Is 8-9.

In the present invention, it is preferable to select the ratio of the number of the shark nin subunit to the target protein in the fusion protein according to the origin of the shark nin used. The ratio of the number of syrup mouth subunits to the number of target proteins (the number of chaperonin subunits: the number of target proteins) can range from 1: 1 to 12: 1, but is preferably 1: 1 to 9: 1. is there. If the number of spore nin subunits per one target protein exceeds 9, it becomes difficult to form spheronin rings.

Specifically, in the case of using bacterial-derived porcine nin, the number of porcine nin subunits: the number of target proteins is 1: 1 or 7: 1 due to the lack of formation of the ring structure of the porcine nin. When a fusion protein is preferred, and archaeal shanin, which comprises eight subunits to form the shanin ring, is used, it is difficult to form a shaunyun ring structure. Ninsubunit number: Target Protein number is preferably 1: 1, 2: 1, 4: 1 or 8: 1. However, depending on the shape and molecular weight of the target protein, a number ratio other than the above may be appropriate. For example, when Escherichia coli-derived protein is used, even if it is a fusion protein in which the ratio of the number of protein subunits to the number of target proteins is 3: 1, two or three molecules of these proteins associate with each other. A ring structure may be formed.

For example, when an archaea-derived scherichia coli having eight subunits constituting a sshaguchi nin ring is used, the number of sshaguchi nin subunits and the number of target proteins are 2: 1. As for the protein, four expressed fusion proteins assemble to form a shaking ring. Number of Shain nin subunits: In a fusion protein with a target protein number of 4: 1, two expressed fusion proteins are assembled to form Shain nin rings.

Therefore, the higher the ratio of the spore mouth ninth submit, the larger the molecular size of the target protein that can be stored in the spheronin cavity. The above purpose In order to avoid the risk that the protein is exposed to the host cytoplasm, it is preferable that there are two or more capronin subunits per target protein.

In the first place, Shapaguchinin not only provides a space separated from the external environment for the target protein, but also has a protein folding function, so that the target protein can be normally folded and its structure is stable. It can also be converted. Normally, the protein folding reaction of chaperonin is performed with a single polypeptide, the substrate protein:

In order to express the function of folding by shanin in the present invention, it is preferable to design a fusion protein such that one target protein is stored in sharonin ring or shanin. However, depending on the molecular weight of the target protein, it can be folded normally even if two or more molecules are stored.

In the fusion protein, the linking pattern between the scharinin subunit and the target protein includes the N-terminal, C-terminal, or scherfet of the spheronin subunit so that the target protein fits within the cavity of the scharinin.ぺ It is preferable to arrange the target protein at the junction between the oral nin subunits. At this time, it is preferable that the chaperonin subunit forms a linked body in which 1 to 20 subunits are linked.

When the target protein has extremely high toxicity to the host or is extremely susceptible to digestion by the host protease, it is preferable to arrange the target protein between the junctions of a plurality of spore nin subunits. Figure 2 shows an example of a fusion protein design using archaeal chaperonin, which has eight submits. According to the present invention, since the target protein expressed as a fusion protein is stored inside the cavity of the Sharp Nin Ring, it is protected from the in vivo environment and is less likely to be digested by the protease. It is preferable that the scallop nin ring further associates to form a non-covalently associated two-layer structure via the ring face.

In addition, even if the target protein has the property of inhibiting important natural mechanisms for the host, since the target protein is separated from the in-vivo environment by Shaun-Yunning, it may interfere with the host's physiological mechanism. No effect is exhibited. In addition, a large number of protein folding intermediates, which are observed when expression is induced by a strong promoter, do not associate with each other. Therefore, expression using a host and inclusion body formation seen in a cell-free translation system can be suppressed. Since shadinin is synthesized into a soluble fraction such as host cytoplasm or body fluid, even if the protein stored inside the shrinin ring is a membrane-bound or transmembrane protein, it is transferred to the membrane. It does not disrupt the host's membrane structure and does not exhibit host toxicity. In addition, if any protein is stored in the same chaperoning ring, it can be purified as a fusion protein under the same purification conditions.

When Shanin is present at a high concentration of 1 ing / mL or more, in the presence of Mg-ATP, the two layers of Sharonin rings are further reversibly bonded via the ring surface to form a fibrous structure. USA, 94, 5383-5388: Furutani, M. eta 1., 1 998 (Trent, JD, etal., 1997, Proc. , J. Biol. Chem. 273, 2839 9_28407). Since the fusion protein of the present invention is formed at a high concentration in a living body, it may form a non-covalently linked fibrous structure via a ring surface or a side surface thereof. Even if the target protein is toxic to the host, storage in the shaved mouth ring is promoted, and high expression of the target protein can be achieved. Even if the fusion protein forms a fibrous structure, it is dissociated for each two-layered ring structure by diluting and reducing the protein concentration, so that the target protein can be recovered.

In the method for producing the protein of the present invention, a gene encoding a fusion protein is prepared using a general genetic engineering technique such as a method using a restriction enzyme or a method using PCR, and an expression vector into which the gene has been introduced is used. The fusion protein can be synthesized in the host.

As the gene encoding the target protein used when producing the gene encoding the fusion protein, a mammalian-derived cDNA or a partial gene encoding an amino acid sequence of 6 or more residues thereof is suitably used.

The host is not particularly limited. For example, bacteria such as Escherichia coli, other prokaryotic cells, yeast, insect cells, animal cells such as mammalian cultured cells, plant cells such as plant cultured cells, animal individuals, and plant individuals And insect individuals. Above all, culture Bacteria or yeast is preferred in terms of low cost, short culture days, simple culturing operation, and the like. In addition, a cell-free translation system using bacteria, eukaryotic extracts, etc. (Spirin, AS, 1991, Science 11, 26561-6266: F alcone, D. eta 1., The fusion protein of the present invention can also be synthesized as a soluble protein by the method of 1991, Mo 1. Cell. Biol. 11, 1, 566-26664).

In general, in Escherichia coli and the like, when the expression plasmid exceeds 10 kbp, the copy number decreases, and as a result, the synthesis amount of the target protein may decrease. For example, in the case of producing a fusion protein in which eight Siaguchi ninth subunits are linked, the expression plasmid is 15 kbp or more. In contrast, the gene encoding the fusion protein is introduced into each of two different plasmids that can coexist and replicate in the same host, and expressed by co-expression in the same host. _c for example it is possible to prevent a decrease in the amount, the same gene that yield the same fusion protein, is introduced into two vectors having different replication regions and drug resistance genes, two drug agents in these two vectors By transforming Escherichia coli or the like in the presence of E. coli and synthesizing a fusion protein, high expression can be obtained.

Further, in the present invention, the gene encoding the fusion protein and the gene encoding only the ninja ninsubunit are introduced into two different plasmids which can coexist and replicate in the same host. Alternatively, they may be co-expressed in the same host. For example, a gene encoding a fusion protein and a gene encoding only a ninnin subunit are introduced into two types of binders having different drug resistance and replication regions, respectively, and are shared in the presence of two types of drugs. The expression can control the structure of Shanin. For example, in the case of producing a fusion protein in which the ratio of the number of Siaguchi nin subunits to the target protein is 4: 1, a vector containing only one or two to four Siaguchi nin subunits is used. By introducing and co-expressing it, it is possible to form a Shaw Nin ring with a 8: 1 ratio between the Scharoen subunit and the target protein. As described above, this method is effective for increasing the expression level, since the enlargement of the plasmid may lead to a decrease in the copy number of the plasmid and the expression level may decrease. The fusion protein gene of the present invention may be introduced into the host chromosome to express the fusion protein without being introduced into the host organism using a vector such as a plasmid. For example, in E. coli, expression of a promoter, a ribosome binding site, a target gene, a terminator, a drug resistance gene, etc. is performed using a site-specific recombination function of lampada integrase in a host that expresses lampada integrase. It is possible to introduce the unit gene into the chromosome (Olson, P. eta 1., 1998, Ρ rotein χ ρ ρ r. Purif. 14, 160-166). In yeast, for example, by homologous recombination using the downstream and upstream sequences of alcohol dextrogenase (AOX) of a methanol-assimilating yeast, the expression unit gene of the target protein including the AOX promoter sequence and the terminator 1 can be obtained. There is a method for integration on the host chromosome (S corer, CA eta 1., 1994, Bio / Techno 1 ogy 12, 2, 181-184). In any case, the expression level can be increased by introducing a plurality of linked expression unit genes into a chromosome. In the expression of a protein by integration of a gene into a chromosome, the expression of a fusion protein having a large molecular weight as in the present invention is stabilized without a decrease in copy number as the size increases, as in the case of using a plasmid. It is possible to do.

Since the fusion protein produced in the present invention has a huge molecular weight of about 650 to 600 kDa, the transcribed mRNA is degraded by a specific ribonuclease, and the translated fusion protein is degraded by a protease. May be cut off in two stages. For example, when E. coli is used as a host, mRNA degradation can be suppressed by using a host deficient in the RNase E gene, a ribonuclease involved in mRNA degradation (G runberg-Manago). , M., 1999, Annu.Rev.Gev., 33, 193-227). To suppress degradation by the prosthesis after translation, expression at a low temperature of 15 to 25 ° C; lon, ompT (Phillipseta 1., 19984, J. B acteriol., 1559, 283-287) ), Clp, Hsl VU (Kanemori, M. eta 1., 1997, J. B acteriol., 179, 721 9). The method used or the like can be used. After synthesizing the fusion protein in the various hosts described above, the cells are collected, disrupted, and the supernatant is collected. Since Schwaen is a giant protein with a molecular weight of about 840-96 OKDa, it can be precipitated by ammonium sulfate salting out at about 40% saturation. After recovering the precipitated protein, the protein is dissolved in an appropriate buffer, and the fraction containing the fusion protein is recovered by hydrophobic chromatography or ion exchange chromatography. After concentrating the recovered fusion protein solution by ultrafiltration, the obtained concentrated solution is treated with about 5 to 50 mM of magnesium chloride and about 50 to 300 mM of sodium chloride or chloride medium. The fusion protein can be purified by performing gel filtration using the contained buffer as a developing solution and collecting the peak immediately after the exclusion limit.

When a tag comprising 6 to 10 histidines is linked to the N-terminal or C-terminal of the fusion protein, the fusion protein can be easily and efficiently recovered using a metal chelate column such as nickel. It can be carried out. In addition, the antibody can be purified quickly and easily by immunoprecipitation or affinity mouth chromatography using an antibody against scharinin or scharonin subunit used. However, in order to recover only the fusion protein having a ring structure, it is preferable to combine them with ion exchange chromatography and gel filtration.

When the Shanin is heat-resistant, most of the E. coli-derived proteins can be precipitated by heat-treating the E. coli extract at 60 to 80 ° C. It can be simplified. In this case, even if the target protein itself is not heat-resistant, it is not denatured because it is retained inside the cavity of Shanin.

Regardless of the purification method using any of the above methods, the morphology of the fusion protein can be observed with a transmission electron microscope, and when the target protein is stored inside the chaperonin ring, the outer diameter is 1 It is possible to observe a ring structure peculiar to Shanin's mouth of about 4 to 16 nm.

In many Chapelön, the association between subunits is stabilized by magnesium ions and ATP. Therefore, when the ring structure of the fusion protein is unstable, it is possible to efficiently recover the fusion protein having the ring structure by allowing magnesium and ATP to be present in the purification process. Meanwhile, get When only the target protein is separated from the obtained fusion protein, the fraction of the fusion protein collected as described above is treated with EDTA (ethylenediaminetetraacetic acid), and then treated with a buffer containing no magnesium or ATP. Dialysis is performed to remove magnesium and ATP. As a result, the interaction between the lip mouth subunits is released, and the tertiary structure of the lip mouth is muddy, exposing the target protein.

In addition, the cleavage sequences of limited proteases such as thrombin, enterokinase, and activated blood coagulation factor 1 ° C are linked to the junction between the ninja ninsubunit and the target protein, and to the ninja subnin subunit. By arranging them at the site, it becomes possible to cut out the target protein from the fusion protein using these limited-degradation-type proteases. In this case, when the fraction of the fusion protein collected as described above is dialyzed, the target protein and the saccharin nin subunit are treated by allowing a limited degradation protease such as thrombin to act on the inner dialysis solution. And can be separated. When the fusion protein of the present invention is used as it is according to the purpose, it is not necessary to intervene a cleavage sequence such as a protease.

After dialysis, high-purity target protein can be easily recovered by subjecting it to ion exchange chromatography, hydrophobic chromatography, or affinity chromatography using an antibody.

When the target protein does not have a methionine residue, the target protein is easily cut out from the spheroid by CNB r by allowing the methionine residue to be present at the junction between the chaperonin subunit and the target protein, Can be released.

If the only purpose is to recover the target protein, the fusion protein does not necessarily need to be purified uniformly.After subjecting the crudely purified sample to EDTA treatment, protease is allowed to act, and the purification procedure is performed according to the target protein. Good. If methionine is not present in the target protein and methionine is present between the chaperonin sap unit and the target protein, the cinnamon ninsubette and the target protein are converted to C N

Since it can be cleaved by Br, the steps of EDTA treatment and dialysis of the fusion protein are not required.

By synthesizing the fusion protein as described above, the soluble fraction in the host cytoplasm A method for producing a recombinant target protein, which produces a fusion protein and extracts only the target protein therefrom, is also one of the present invention.

When the target protein is a membrane-bound protein or a transmembrane protein, the target protein may be insolubilized by separating the target protein from the ninnin unit, but in this case, only the insoluble material is removed. After recovery by centrifugation, if a non-ionic surfactant with a hydrophobic alkyl chain length of about octyl (8 carbon atoms) to dodecyl (12 carbon atoms) is used, the micelle diameter will be almost It is easy to solubilize according to its thickness. Examples of such a nonionic surfactant include β-otatildanorecoside, Triton X-100, Nonidet P-40, Tween 20, and the like.

According to the present invention, expression of toxicity of a target protein to a host, degradation by a protease, and formation of an inclusion body are ensured by assembling the target protein as a fusion protein with a porcine nin in the cavity of the spheronin ring. By solving this problem, it can be expressed in large amounts as a soluble protein. Further, purification can be performed efficiently. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to only these Examples.

[Example 1]

(ThermococcusKS KS—synthesis of 1 strain spliced j3 submit conjugate)

The Shanin nin iS subunit (TCP) 3 gene shown in SEQ ID NO: 1 was cloned by PCR (Po1 ym erase chain reaction) using Thermococcus KS-1 strain genome as type III. An expression vector with a 7 promoter into which a gene fragment in which the TCP β gene is linked one, two, three and four times in one direction! ) We constructed ETD (TCP J3) η (η is 1 to 4) (Fig. 3). Each expression vector was introduced into Escherichia coli BL21 (DE3) strain, and XY. Medium (16 g of pactotryptone, 10 g of yeast extract, 15 g of NaC at 15 g ^/ L) at 30 ° C for 24 hours Then, a conjugate of the Shahkou nin subunit was expressed. After completion of the culture, the collected cells were disrupted by sonication, the supernatant was recovered by centrifugation, and analyzed by SDS-PAGE (Fig. 4). From the results of SDS-PAGE, it was confirmed that (TCP) 3) n (n is 1 to 4) was overexpressed in the soluble fraction of the cytoplasm.

[Example 2]

(Observation of T CP ^ linked body by transmission electron microscope)

Cleavage of pETD (TCPβ) 2 and pETD (TCPj3) 4 with M1uI followed by self-ligation yields the C-terminal of TCP / 3 dimer and TCPβ tetramer. Thus, expression vectors p ETDH (TC P jS) 2 and p ETDH (TC P / 3) 4 for synthesizing a recombinant protein having 6 residues of histidine added thereto were obtained (see FIG. 3). After transforming the BL21 (DE3) strain with this vector, an Escherichia coli extract in which a Shapachinin j3 submit conjugate was expressed was obtained under the same conditions as in Example 1. The cell extract was subjected to heat treatment at 75 ° C for 30 minutes at a protein concentration of 5 mgZmL to denature and precipitate most of the E. coli-derived proteins. The supernatant was recovered by centrifugation and applied to a nickel chelate Sepharose column. 1 After thoroughly washing the column with 50 mM Na-phosphate buffer (pH 7.0) containing OmM imidazole, transfer to nickel-chelated sepharose with the same buffer containing 500 mM imidazole. The adsorbed fraction was eluted. As a result of confirming the eluted fraction by SDS-PAGE, it was found that TCP i3 dimer and TCP tetramer were recovered. The obtained fraction was dialyzed against 2 5mM T ris- HC 1 buffer containing _{5mM Mg C 1 2 (p H} 7. 5), the internal dialysate T SKg el S uper Q- 5 PW column Separation was performed by anion exchange chromatography using a Toso (manufactured by Toso), and the TCPβ dimer and TC TCβ tetramer were uniformly purified.

Each purified sample was negatively stained with 0.2% peranyl acetate, and the morphology was observed with a transmission electron microscope. A ring structure unique to nin was formed. from this result, It was found that even if TPPβ was connected between the subunits, each molecule was aggregated to form a ring structure unique to Shadinin. It is considered that four molecules of TCP dimer assemble to form one ring, and that of TCP 34-mer as two molecules to form one ring.

[Example 3]

(Synthesis of fusion protein of TCP i34mer and HBs antigen)

For the hepatitis B virus surface antigen (HBs antigen) gene shown in SEQ ID NO: 2, a SpeI site was set at the 5 'end and an HpaI site was set at the 3' end by PCR. And HpaI treated; to synthesize a fusion protein of pETDH (TCP) 4 and a fusion protein of TCP | 34-mer and HBs antigen with 6-residue histidine at the C-terminus. An expression vector pETDH (TCP J3) 4 · HBs was constructed. After transforming the BL21 (DE3) strain with this vector, a fusion protein was synthesized under the same conditions as in Example 1. After separating the soluble fraction of Escherichia coli by SDS-PAGE and analyzing by Coomassie propriant blue staining, a band corresponding to the size of the fusion protein was detected (Fig. 4). After SDS-PAGE, the DNA was transferred to a blotting membrane and subjected to Western plotting using an anti-HBs antigen polyclonal antibody. As a result, an E. coli extract expressing only TCP; 34-mer was negative. However, a positive band corresponding to the size (about 260 KDa) was detected only in the extract of E. coli in which the fusion protein was synthesized. This indicates that the HBs antigen is expressed in the soluble fraction of Escherichia coli as a fusion protein with the TCP] 34-mer. In the expression experiment using the HBs antigen alone, the soluble fraction and the precipitated fraction of Escherichia coli were negative by the same Western plotting. (Purification of recombinant HB s antigen)

In the same manner as in Example 2, the fusion protein was recovered by nickel chelate column after removal of the imidazole by dialysis, § by TSKg el S uper Q- 5 PW column Ru using the eluent containing 5 mM Mg C 1 ₂ - The fusion protein of the TCP | 34mer and the HBs antigen was purified by on-exchange chromatography. Also, The presence of HBs antigen was confirmed by Western plotting using an anti-HBs antigen polyclonal antibody. Observation of the obtained fusion protein with a transmission electron microscope revealed that it had formed a ring structure unique to Shadinin. This suggested that two molecules of the fusion protein were assembled to form a ring structure. The collected fraction was incubated in the presence of ImM EDTA-2 Na (ethylenediaminetetraacetate), treated with PreScissionprotease (Amersham-Pharmacia Biotech) at 4 ° C. In—incubated day and night. The resulting insolubles were recovered by centrifugation and then dissolved in 1.0%; 3-octyldarcoside. The HB s antigen in the obtained lysate was detected by an EIA kit for measurement of HB s antigen, "Enzygnost-1 HB s Ag monoc 1 onal" (manufactured by Hoechst-Bering GmbH). Further, as a result of analysis by Western blotting, a band having a molecular weight corresponding to HBs antigen of about 25 kDa was specifically detected. From the above, it was found that the recombinant HBsAg could be excised from Shanin by limited protease. Further, it was estimated that about 40 mg of HBsAg was expressed in the soluble fraction per 1 L of E. coli culture by the expression method of this example.

[Example 4]

(Co-expression of a fusion protein with a 2: 1 ratio of the number of shaperonin subunits to the number of HB s antigens and the double linking of the shadinin J3 subunit)

An expression unit of (TCPNβ) 2 containing a T7 promoter was recovered from BET1 (TC PN / 3) 2 (ampicillin resistance) prepared in Example 2 by cleavage with Bg1II and NotI. This was cloned into pACYC184 plasmid (Nippon Gene) to construct pATH (TCPNi3) 2 (chloramphenecol resistant). Transform E. coli with p ETDH (TC PN j3) 2 and pATH (TCPN) 3 on LB agar medium containing ampicillin (100 μg / mL) and chloramphenicol (15 μg / mL) The grown 10 colonies were inoculated into 2X YT liquid medium (16 g of batatotryptone, 10 g of yeast extract, and 15 g / L of NaC), and ampicillin (100 / x gZmL) and chloramfeucol (3 (4 μg / mL) at 30 ° C and overnight.

As a result of confirming protein expression from the obtained cells by SDS-PAGE, a dimer of about 145 kDa fusion protein and about 120 kDa shadinin β-subunit was expressed. That was confirmed. In addition, Western blotting using an anti-HBs antigen polyclonal antibody detected only a band equivalent to 145 KDa. A fraction containing the fusion protein was collected from the E. coli extract using an Eckerl chelate column as in Example 2. Further, after removing imidazole by dialysis, an aion exchange chromatography using a TSKgel SuperQ-5 PW column was performed to purify a fraction containing the fusion protein. Observation of the obtained protein with a transmission electron microscope revealed that it had formed a ring structure unique to shahkonin. As a control, when only the fusion protein in which the ratio of the number of subunits to the number of 3 subunits to the number of HBs antigens was 2: 1 was expressed, the SDS-PAGE and Western plotting results It was determined that the expression level was much lower than in the expression method. From the above, the fusion protein with a ratio of 2: 1 between the number of subunits and the number of HB s antigens and the double-linkage of subunits with 3 subunits form a ring structure by assembling each other. HBsAg can be expressed in large amounts by forming and storing the HBsAg inside the cavity.However, with the fusion protein alone, it is difficult to form a ring structure due to steric hindrance. It is considered that the expression was suppressed. According to the expression method of this example, it was estimated that about 70 mg of HBs antigen was expressed in the soluble fraction per 1 L of E. coli culture solution. The expression level of the expression method of this example was higher than that of the fusion protein synthesis of TCP tetramer and HBs antigen (Example 3).

[Example 5]

(Synthesis of fusion protein of T C Ρ] 34-mer and HCV core antigen)

For the hepatitis C virus core antigen (HCV c antigen) gene shown in SEQ ID NO: 3, a SpeI site was set at the 5 'end and an HpaI site was set at the 3' end by PCR. introduced into p ETDH (TCP) 4 that I and Hp a I-processed, TCP beta 4 expression vectors for the synthesis of a fusion protein with dimer and HCV c antigen _Ρ ETDH (TCP) 3) 4-HCV c was constructed. After transforming the BL21 (DE3) strain with this vector, a fusion protein was synthesized under the same conditions as in Example 1. After separating the soluble fraction of the E. coli crushed product by SDS-PAGE and analyzing it by Coomassie-Prirant blue staining, a band corresponding to the fusion protein was detected (Fig. 4). After SDS-PAGE, the DNA was transcribed into a plotting membrane plan and subjected to Western plotting using an anti-HCV c antigen monoclonal antibody. As a result, an E. coli extract expressing only the TCP tetramer was negative. However, in the E. coli extract from which the fusion protein was synthesized, a positive band corresponding to its size (about 26 OKD a) was detected (Fig. 6). This indicated that the HCVc antigen was expressed in the soluble fraction of Escherichia coli as a fusion protein with the TCP tetramer. As a result of performing an expression experiment using HC Vc antigen alone as a control, the precipitated fraction of Escherichia coli was positive by the same Western plotting, but the soluble fraction was negative. This indicates that the HCVc antigen alone is all expressed as an inclusion body, but can be expressed in the soluble fraction as a fusion protein with the Sharp mouth subunit tetramer. The fusion protein was purified using a nickel chelate column and a TSKgel Super Q_5 PW column in the same manner as in Example 3. Observation of the obtained fusion protein with a transmission electron microscope revealed that it had formed a ring structure unique to Shaguchinin. This suggests that two molecules of the fusion protein aggregate to form a ring structure. The collected fraction was incubated in the presence of ImM EDTA-2Na, and then dialyzed against a 50 mM K-phosphate buffer (pH 7.0). During dialysis, Prescissionprotease (Amersham 'Pharmacia' Biotech) was allowed to act on it overnight, followed by incubating overnight at 4 ° C. Thereafter, the reaction solution was fractionated by a T SKgel Super Q-5 PW column. After coating the protein of each fraction on a 96-well microtiter plate, blocking was performed with bovine serum albumin, and PBS-T buffer (10 mM Na-phosphate buffer pH 7.5, 0.8% Washed three times with sodium chloride, 0.05% Tween 20). Next, human positive serum or human negative serum diluted with PBS-T buffer was added and reacted. After washing with PBS-T buffer, a peroxidase-labeled human IgG antibody was allowed to act. After the reaction is complete, use PBS-T After washing four times, a substrate color developing solution containing furdiamine and hydrogen peroxide was added and reacted. After stopping the reaction by adding 4 N sulfuric acid, the absorption at 490 nm was measured. Analysis of the detected HCV c antigen-positive fraction by SDS-PAGE revealed that almost uniform HCV c antigen of approximately 22 kDa was purified. From the above, it was found that the recombinant HCV c antigen can be excised from Shanin by a limited protease. Further, it was estimated that about 80 mg of HCV c antigen was expressed in the soluble fraction per 1 L of E. coli culture by the expression method of this example. [Example 6]

(Synthesis of fusion protein between TCP β tetramer and anti-lysozyme sc FV antibody) For the mouse-derived anti- ヮ trilysozyme single chain antibody (anti-HEL-singlechain Fv antibody: Hsc FV) gene shown in SEQ ID NO: 4 Then, by PCR, a SpeI site was provided at the 5 'end and an HpaI site was provided at the 3' end, and pETDH (TCP] 3) 4 treated with SpeI and HpaI was used. Then, an expression vector p ETDH (TCP j3) 4 ■ H sc FV for synthesizing a fusion protein of the TCP | 34 tetramer and H sc FV was constructed. After transforming the BL21 (DE3) strain with this vector, a fusion protein was synthesized under the same conditions as in Example 1. The soluble fraction of the disrupted E. coli was separated by SDS-PAGE and analyzed by Coomassie Brilliant Blue staining. As a result, a band having a size corresponding to the fusion protein was detected. After SDS-PAGE, the DNA was transferred to a blotting membrane and subjected to Western plotting using an anti-6HIs monoclonal antibody, an antibody that recognizes six histidine residues. A positive band corresponding to the size (about 265 KDa) was detected in the E. coli extract in which the fusion protein was synthesized, while the E. coli extract in which only the monomer was expressed was negative. This indicated that Hsc FV was expressed in the soluble fraction of Escherichia coli as a fusion protein with the TCP ^ tetramer. As a result of an expression experiment using Hsc FV alone as a control, the precipitated fraction of E. coli was positive by the same Western plotting, but the soluble fraction was negative. From this, all of Hsc FV alone are expressed as inclusion bodies. However, it was found that it can be expressed in the soluble fraction as a fusion protein with the Shaman nin subunit tetramer. Also, it was estimated that about 75 mg of Hsc FV was expressed in the soluble fraction per 1 L of E. coli culture by the expression method of this example.

[Example 7]

(Synthesis of fusion protein of TCP β tetramer and human-derived antibody heavy chain constant region) The human-derived antibody heavy chain constant region (AbHC) An SpeI site at the end and an HpaI site at the 3 'end were introduced into pETDH (TCP) 3) 4 treated with SpeI and HpaI, and a TCP β tetramer was introduced. An expression vector pETDH (TCPβ) 4 4AbHC for synthesizing a fusion protein with AbHC was constructed. After transforming the BL21 (DE3) strain with this vector, a fusion protein was synthesized under the same conditions as in Example 1. After separating the soluble fraction of Escherichia coli by SDS-PAGE and analyzing by Coomassie propriant blue staining, a band corresponding to the fusion protein was detected (Fig. 4). In addition, after SDS-PAGE, the results were transferred to a plotting membrane and subjected to pattern plotting using an anti-human IgG_Fc antibody, which is an antibody that recognizes the Fc region of a human-derived antibody. However, a positive band corresponding to the size (about 270 KDa) was found in the E. coli extract in which only the TCP tetramer was expressed, while the E. coli extract in which the fusion protein was synthesized had a negative value. Detected (Figure 7). This indicated that Ab HC was expressed in the soluble fraction of Escherichia coli as a fusion protein with TCP <34 tetramer. As a result of performing an expression experiment using Ab HC alone as a control, both the soluble fraction and the precipitated fraction of E. coli were negative in the same Western plotting. From this, it was found that Ab HC alone was hardly expressed in Escherichia coli, but could be expressed in the soluble fraction as a fusion protein with the Shadin nin subunit tetramer. In addition, it was estimated that about 75 mg of AbHC was expressed in the soluble fraction per liter of the E. coli culture solution by the expression method of this example. [Example 8]

(Expression of Escherichia coli Shanin N-GroEL conjugate)

The Escherichia coli Shaguchinin GroEL gene shown in SEQ ID NO: 6 was cloned by PCR using the Escherichia coli K12 strain genomic as type III. An expression vector pT r (Gro E) n (n is an expression vector having a trc promoter into which a gene fragment in which the Gro EL gene is linked in one direction 1, 2, 3, 4, 5, 6, and 7 times is inserted. 1 to 7) (Fig. 8). Each expression vector was introduced into Escherichia coli BL 21 (DE 3) strain, and 2XY.T. medium containing carbenicillin (100 g / mL) (batat tryptone 16 g, yeast extract 10 g, NaC 15 g) / L) at 25 ° C for 24 hours to express a lipase-subunit conjugate. After completion of the culture, the cells were collected and disrupted by ultrasonication. After collecting the supernatant by centrifugation and analyzing by SDS-PAGE, it was confirmed that (GroE) n (n is 1 to 7) was overexpressed in the soluble fraction (Figure 9). . Recombinant (GroE) 7 was purified from the recovered E. coli extract by DEAE-Sepharose, TSKgelSuperQ-5PW and gel filtration. As a result of observing the obtained purified sample with a transmission electron microscope, a ring structure peculiar to Shaguchinin was observed. From this, it was found that the Escherichia coli Shaguchinin GroEL maintains the 7-fold rotationally symmetric structure even when all the subunits are connected.

[Example 9]

(Synthesis of fusion protein of Escherichia coli Shanin N Gro 7 times conjugate and human interferon)

For the human interferon α2b (INF) gene shown in SEQ ID NO: 7, a NheI site was provided at the 5 ′ end and an XhoI site was provided at the 3 ′ end by PCR, and NheI and An expression vector for synthesizing a fusion protein of a GrO EL 7-fold conjugate and INF by introducing it into pTr (GroE) 7 that has been treated with XhoI: pTr (GroE ) 7 · INF was constructed. After transforming this expression vector into Escherichia coli strain BL21 (DE3), a fusion protein was synthesized under the same conditions as in Example 8. Expression using pTr (GroE) 7 as a control and expression of INF alone were also performed. After separating the supernatant and precipitate fraction of each E. coli extract by SDS-PAGE, The DNA was transferred to a mouth setting membrane and subjected to Western blotting using an anti-INF polyclonal antibody. As a result, only the E. coli extract sample having pT r (G ro E) Ί · I NF showed a strong band in the soluble fraction at a position corresponding to the molecular weight of the fusion protein (250 to 26 OD a). It was found that most of the expression of INF alone was produced in the insoluble fraction. From the above, it was found that IFN was expressed as a soluble protein when expressed as a fusion protein with the Escherichia coli Gro EL seven-fold conjugate. Salting out of E. coli extract containing pT r (Gro E) 7 'I NF, anion exchange chromatography using DEAE-Sepharose and TSKge 1 Super Q-5 PW columns, and Superose 6 (Amersham) ■ The fusion protein was purified by gel filtration using Pharmacia (Biotech). As a result of observing the obtained purified sample with a transmission electron microscope, a ring structure peculiar to Shaguchinin was observed. From the above, it is considered that INF was expressed in the soluble fraction by being stored for each molecule inside the cavities of the Gro EL.

[Example 10]

(Synthesis of fusion protein of Escherichia coli Shanin N Gro 7 times conjugate and serotonin receptor)

For the human serotonin receptor (5HT1A) gene shown in SEQ ID NO: 8, a NheI site was set at the 5 'end and a XhoI site at the 3' end by PCR, and NheI and XhoI An expression vector pTr (GroE) 7 which is introduced into the treated pTr (GroE) 7 and synthesizes a fusion protein of 7HT-linked GroEL and 5HT1A. ■ 5HT1A was constructed. After transforming this vector into Escherichia coli BL21 (DE3), a fusion protein was synthesized under the same conditions as in Example 8. As a control, expression using pT r (GroE) 7 and expression of 5HT 1A alone were also performed. After separating the supernatant and the precipitated fraction of each E. coli extract by SDS-PAGE, they were transferred to a blotting membrane, and subjected to Western blotting using an anti-5HT1A polyclonal antibody. As a result, a soluble fraction was obtained from only the E. coli extract sample containing T r (Gro E) 7 ■ 5HT1A. A strong band was detected at a position corresponding to the molecular weight of the synthesized protein (about 280 kDa). No significant size band was detected in the soluble or insoluble fractions in the expression of 5HT1A alone. From the above, it was found that 5HT1A cannot be expressed alone in Escherichia coli, but can be expressed as a soluble protein by expressing it as a fusion protein with the GroEL seven-fold conjugate. p T r (G ro E) 7 ■ Salt extraction, Anion exchange chromatography using DEAE-Sepharose and TSK ge 1 Super Q-5 PW "columns, and S The fusion protein was purified by gel filtration using uperose 6 (Amersham 'Pharmacia' Biotech), and the resulting purified sample was observed with a transmission electron microscope, revealing a ring structure unique to Sharpinin. Based on the above, it is considered that 5HT 1A was synthesized into a soluble fraction by being stored in a molecule of the Gro EL for each molecule.

[Example 11]

(Synthesis of fusion protein between (TCP) 4 and HBs antigen by cell-free translation system) For cell-free translation, expression containing a gene encoding a fusion protein between TCP 34-fold conjugate and HBs antigen The vector pIV (TCP | 3) 4'HBs was constructed. The reaction is usually carried out by adding pIV (TCPβ) 4HBS to a reaction solution containing RNA polymerase, ribosomes, amino acids, nucleotides, aminoacyl-tRNA synthetase, etc. This was performed by incubating with. After completion of the reaction, the reaction solution was purified to a single protein by nickel chelate chromatography and a Tskgel Super Q-5 PW column. Observation of the purified fusion protein with a transmission electron microscope revealed a ring structure unique to Shahkonin. The HBs antigen was excised from the purified fusion protein using PreScissionprotease in the same manner as in Example 3, and then the insoluble HBs antigen was solubilized by octyldarcoside by centrifugation. After subjecting this sample to SDS-PAGE and performing Western plotting with an anti-HBs antigen polyclonal antibody, a band of about 25 KDa corresponding to the molecular weight of the HBs antigen was detected. Similarly insoluble when expressed with HBsAg alone Although HBs antigen accumulated in the fraction, it was difficult to solubilize it with one-year-old octyldarcoside. As described above, the synthesis of HBs antigen using a fusion protein with (TCP j3) 4 was also effective in a cell-free translation system. [Example 12]

(Synthesis of fusion protein of (G ro E) 7 and 5 HT 1 A by cell-free translation system) In the same manner as in Example 11, a fusion protein of a G ro EL 7-fold conjugate and 5 HT 1 A Quality cell-free synthesis was performed. As a control, 5HT1A alone was also synthesized. After completion of the reaction, Western blotting was performed using an anti-5HT1A polyclonal antibody.As a result, a band corresponding to the molecular weight of the fusion protein (about 280 KDa) was detected in the soluble fraction of the fusion protein. . After purifying the fusion protein in the same manner as in Example 11, observation with a transmission electron microscope revealed a ring structure unique to chaperonin. In the synthesis of 5HT1A alone, it was detected only in the insoluble fraction. Based on the above, 5HT1A alone is expressed as an insoluble protein in a cell-free translation system, but when expressed as a fusion protein with the Gro EL 7-fold conjugate per molecule, the fusion protein is absent. The protein was also synthesized as a soluble protein in the cell translation system. Industrial applicability

Since the method for producing a protein and the fusion protein of the present invention have the above-mentioned constitutions, they are useful for increasing the amount of synthesis of a protein that is difficult to express in large amounts and a recombinant protein that is difficult to express in a soluble fraction. It is.

Claims

The scope of the claims

1. Transcribe and translate the gene containing the gene encoding the Siaguchi ninsubunit and the gene encoding the protein of interest, and the target protein is linked to the Siaguchi ninsubunit via a peptide bond. A method for producing a protein, comprising synthesizing a fusion protein.

2. The fusion protein is composed of 1 to 20 linked Siaguchi nin subunits, the N-terminus of the linked Siaguchi sapunit, the C terminus of the linked Siaguchi ninsubunit, or the Sshaguchi. 2. The method for producing a protein according to claim 1, comprising a target protein linked via a peptide bond to a link between the nin subunits.

3. A gene containing the gene encoding the ninja ninsubunit and the gene encoding the target protein are introduced into each of two different plasmids that can coexist and replicate in the same host, and 3. The method for producing a protein according to claim 1, wherein the protein is co-expressed in a host.

4. It is possible to coexist and replicate in the same host, a gene containing a gene encoding a spore mouth nin subunit and a gene containing a gene encoding a target protein, and a gene coding only a sham mouth nin. 3. The method for producing a protein according to claim 1, wherein the protein is introduced into two different plasmids and co-expressed in the same host.

5. The fusion protein according to claim 1, wherein the target protein is stored in a chaperonin ring in a state where the target protein is linked via a peptide bond to a gamma nin subunit. 5. The method for producing a protein according to claim 2, 3, 3 or 4.

6. Sharp Nin Ring is a non-covalently associated two-layer structure via the ring face 6. The method for producing a protein according to claim 5, wherein the protein is formed.

7. The method for producing a protein according to claim 5, wherein the scallop nin ring forms a fibrous structure which is non-covalently linked via a ring surface or a side surface thereof.

8. A step of providing a cleavage sequence for a limited-degraded protease at the junction between the lip mouth protein and the target protein, and cleaving the target protein from the fusion protein with the limited-degraded protease. 8. The method for producing a protein according to the first, second, third, fourth, fifth, sixth or seventh range.

9. The method for producing a protein according to claim 8, wherein a cleavage sequence for a limited-degradation type protease is provided at a connecting portion between the ninth ninsubunits.

10. The method according to claim 1, further comprising the step of: providing a methionine residue at a junction between the ninth subunit and the target protein, and cleaving the target protein from the fusion protein with CNBr. 8. The method for producing a protein according to claim 3, 3, 4, 5, 6, or 7.

11. The claim 1, 2, 3, 4, 5, 6, 7, 8, 9, or 1 characterized in that the organism from which Shanin is derived is a bacterium, archaea or eukaryote. The method for producing a protein according to claim 0.

12. The claim 1, characterized in that the fusion protein is synthesized by a host of any of a bacterium, a yeast, an animal cell, a plant cell, an insect cell, an animal individual, a plant individual, or an insect individual. 2. The method for producing a protein according to 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11.

13. The first claim, wherein the fusion protein is synthesized in a cell-free translation system. 2. The method for producing a protein according to 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11.

14. The gene encoding the target protein, wherein the mammal-derived cDNA or a partial gene encoding an amino acid sequence of 6 or more residues of the mammalian-derived cDNA, 2. The method for producing a protein according to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 11, 12 or 13.

1 5. The target protein is the heavy chain of a mammal-derived antibody, the light chain of a mammal-derived antibody, or the full-length FV region single-chain antibody of a mammal-derived antibody, or at least six residues thereof. The protein according to claim 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10, 11, 12, 13, or 14, wherein the protein is a partial protein. Production method.

1 6. The target protein is a viral antigen, a seven-transmembrane receptor protein, or a cytokinin, wherein the first, second, third, fourth, fifth, sixth, seventh, and seventh claim 8. The method for producing a protein according to 8, 9, 10, 11, 12, or 13.

1 7. A fusion protein consisting of a Shanin ninsubunit and a target protein, wherein the target protein is connected to the Sharonin subunit via a peptide bond, and A fusion protein characterized by being stored.

18. The fusion protein according to claim 17, wherein the ninth ring has a two-layer structure non-covalently associated via a ring surface.

19. The fusion protein according to claim 17, wherein the mouth opening ring forms a fibrous structure linked non-covalently via the ring surface or the side surface thereof. .